Overview of HIV-1 Resistance Assay Methods

Resistance assays use different technologies that provide complementary information about antiretroviral resistance. The 2 different types of drug resistance tests available are assays for genotype and phenotype.^[1,2] Genotype assays provide information about viral mutations that may result in changes in viral susceptibility to particular drugs or classes of drugs. Phenotype assays directly quantitate the level of susceptibility of a patient's virus sample to specific drugs in vitro. The values measured from the patient sample are compared with values measured from a standard wild-type (WT) reference strain. The degree of phenotypic resistance is the difference in susceptibility to a particular drug between the patient sample and the reference strain. Both genotype and phenotype testing methods require the use of polymerase chain reaction (PCR) technology to amplify the HIV-1 genes of interest (PR and RT) from patients' plasma samples. However, there are numerousdifferences between these 2 resistance testing methods. One methodologic approach, the other, or both may be preferable in certain situations.^[1,2]

Genotype

Genotypic resistance assays use state-of-the-art DNA sequencing methods to examine the RT and PR regions of the HIV-1 genome for all possible resistance-associated mutations. However, some genotypic methods (line-probe assay or chip-based) interrogate only a limited subset of the positions in which resistance mutations may occur; these limited tests may be less appropriate for clinical use as an adjunct to managing the use of various antiretroviral combinations.^[1,2] They do have some advantages over sequencing assays that may lead to a useful clinical niche with further development, however: they are cheaper, faster, and better able to detect minority subpopulations (to 10% or less). These features may prove advantageous for use of a line probe assay in screening, for example, in certain clinical settings.

The major limitation of genotype assays lies in the clinical interpretation of the detected mutation patterns, given the complexity of available data and the ongoing acquisition of new data (discussed further below).

The results of a genotype assay are reported in the form of the resistance mutations identified, described above in the section "How Mutations Are Designated." Some test reports may also include an interpretation of the degree of resistance conferred by the particular mutation pattern to certain drugs based on published data or presented as in vitro and/or in vivo data.

Viral genome sequencing can be performed using kits such as the TRUGENE HIV-1 Genotyping Kit, by Visible Genetics, Inc. (VGI), or the ViroSeq HIV-1 Genotyping System, by Applied Biosystems, Inc. (ABI). Some testing laboratories have developed their own assays, which are referred to as "home-brew" assays. The TRUGENE HIV-1 Genotyping Kit became the first kit to be approved by the Food and Drug Administration (FDA), in September 2001.

The retail cost of a genotype assay is approximately $400-$500 per sample, and results are generated in 1-4 weeks, with most laboratories providing results in 2 weeks.^[1-3] It is likely that most clinical laboratories offering viral load testing will eventually adopt FDA-approved resistance tests, which may eliminate or reduce the time required for shipping and processing of specimens. As a result, a significant improvement in reporting time and the possibility of local specimen testing, including local prioritization of certain specimens (eg, needle-stick source specimens), should make genotypic resistance testing more timely and affordable for patient management. The use of FDA-approved kits should also provide an advantage in improving standardization and consistency of mutation detection and interpretation.

Phenotype

Phenotypic assays use recombinant virus composed of a patient's virus PR and RT genes, which are inserted into a standard reference strain of virus. The recombinant virus is then tested in vitro for the amount of each particular drug needed to inhibit virus replication by 50% (50% inhibitory concentration, IC₅₀), relative to the amount of drug needed to inhibit a reference strain of virus. Results are reported in the form of the fold change in drug susceptibility relative to a reference strain of HIV-1 (Figure 2).^[1,2] An increase in IC₅₀, increase in resistance, and decrease in susceptibility are each different ways of expressing the fact that more drug is needed to inhibit the patient's virus in vitro. relative to the reference virus. Two recombinant virus phenotypic assays are available from commercial laboratories in the United States: PhenoSense HIV from ViroLogic, Inc. (South San Francisco) and Antivirogram from Tibotec-Virco GroupNV (Belgium). A third assay is commercially available in Europe.

While genotypic assays search for mutations in the viral genome that may have an impact on viral susceptibility to certain drugs, phenotypic assays directly measure the impact of various drug concentrations on viral replication. Figure 2 shows the relationship between the percent inhibition of viral replication (Y-axis) of WT and mutant strains of HIV and increasing drug concentrations (X-axis). The shift to the right of the sigmoidal concentration curve of the mutant HIV strain illustrates that greater concentrations of drug are needed (IC₅₀) in order to provide the same level of inhibition relative to the WT strain. The major limitation now is in clinical interpretation of an IC₅₀: what fold change in resistance predicts escape from drug inhibition in vivo. This is still under study and varies by drug, with data now available for only a few drugs. The "clinical cut-off" for an IC₅₀ is also probably best used if it can be related to interpatientdifferences in blood levels of the drug in question (discussed further below).